2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system

We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells, DESC was the most potent antagonist; of spermine transport in ZR-75-1 human breast cancer cells, with K-i values of 5.0 +/- 0.7, 80 +/- 31, and 16 +/- 3 mu m for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (K-i = 1.6 +/- 0.5 and 2.7 +/- 1.1 mu M, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/ noncompetitive antagonist, Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low K-i toward polyamine transport, The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [H-3]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation Moreover, as little as 0.3 mu M spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 mu M DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a patent competitive inhibitor mag be kinetically inadequate to block; replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.